Method of concentrating and separating dopaminergic neurons

ABSTRACT

The invention of this application provides a method comprising introducing a reporter nucleic acid molecule that expresses a fluorescent protein under control of the promoter/enhancer of a gene that is expressed in dopaminergic neurons, into each of cells, and isolating fluorescence-emitting cells. The invention also provides a method for visualizing and identifying dopaminergic neurons alive that exist with in cells, which comprises introducing the above-mentioned reporter nucleic acid molecule into each of cells, and measuring the fluorescence distribution within the cells. The invention further provides a method for identifying a dopaminergic neurons-inducing factor, which comprises introducing the reporter nucleic acid molecule into cells that have the ability to differentiate into dopaminergic neurons, then incubating the cells with a candidate substance, and determining whether the candidate substance is a dopaminergic neurons-inducing factor by using the fluorescence of the cells as an indicator.

This application is a 371 of PCT/JP00/08674 filed Dec. 7, 2000.

TECHNICAL FIELD

The invention of this application relates to a method for enrichmentand/or isolation of dopaminergic neurons. More precisely, it relates toan efficient and reliable method of identifying dopaminergic neurons forenriching and/or isolating them, in which the dopaminergic neurons areuseful as graft cell for treatment of Parkinson's disease and asmaterials for developing methods of treating the disease.

BACKGROUND ART

Parkinson's disease is caused by selective degeneration and deletion ofdopaminergic neurons in mesencephalic substantia nigra. For treating it,the effectiveness of transplanting a fetal mesencephalon tissue thatcontains a large amount of dopaminergic neurons (or cells having thedifferentiation potency into dopaminergic neurons), into the brain(striate body) of patients has been verified.

In fact, however, it is impossible to secure plenty of fetal braintissue for use in ordinary clinics. Therefore, donor cells substitutablefor fetal mesencephalon are desired.

For example, it is under investigation to use cells that have beendifferentiated into dopaminergic neurons from a large number ofundifferentiated neural cells, for donor cells for transplantation. Inaddition, it is also under investigation to use cells having beendifferentiated from non-neural cells such as ES cells or marrowmesenchymal cells into dopaminergic neurons, for donor cells fortransplantation. These cells can be differentiated into the intendeddopaminergic neurons, after in vitro expansion, and therefore could be ameans for solving the problem of shortage of donors. Moreover, sincemarrow mesenchymal cells can be safely collected from adults, it ispossible to prepare dopaminergic neurons for transplantation from thecells of patients themselves. Accordingly, if those kinds of therapeuticstrategy become available, they will solve not only the problem ofshortage of donors and the technical problem of rejection againstgrafts, but also the ethical problem involved in obtaining dopaminergicneurons from aborted babies.

However, the method for the efficient induction of dopaminergic neuronsfrom undifferentiated cell groups is not as yet completely established.In addition, from undifferentiated cell groups, various cells other thandopaminergic neurons are differentiated. Further, there is a risk thatundifferentiated cell groups may include cells that will form tumorsafter transplanted. Accordingly, if dopaminergic neurons that had beendifferentiated in vitro are intended to be used for transplantation,they must be selectively separated from many kinds of cell groups.

As so mentioned hereinabove, enriched dopaminergic neurons are expectedto be useful for graft donor cells for treatment of Parkinson's disease,etc. In addition, the technique of enrichment and/or isolation ofdopaminergic neurons is extremely useful for identifying novel proteinsand genes that are expressed specifically in these neurons. This isbecause such proteins and genes are expected to lead to novel drugs forthe treatment.

In addition, it is extremely important to identify the factor thatinduces the in vitro differentiation of dopaminergic neurons fromundifferentiated cells. Not only the factor is useful for efficientlyinducing dopaminergic neurons from undifferentiated cells, but also thefactor itself is expected to lead to novel drugs for the treatment.

As yet, however, no method has been established for isolatingdopaminergic neurons from in vivo tissues or from cells being culturedin vitro.

Needless-to-say, not only the method for searching for the factor ofin-vitro induction of dopaminergic neurons but also the method necessaryfor the search, which is for visualizing living dopaminergic neurons,has not been established as yet.

One object of the invention of this application is to provide a methodfor visualizing living dopaminergic neurons in cells including varioustypes of different cells, to thereby enrich and isolate the dopaminergicneurons to a high purity.

Another object of the invention of this application is to provide thedopaminergic neurons isolated by the method.

Still another object of the invention of this application is to providea method for identifying a factor that induces the differentiation ofdopaminergic neurons from undifferentiated cells.

DISCLOSURE OF THE INVENTION

In its first aspect, the invention of this application provides a methodfor enrichment and/or isolation of dopaminergic neurons from cells,which comprises introducing a reporter nucleic acid molecule thatexpresses a fluorescent protein under control of the promoter/enhancerof a gene that is expressed in dopaminergic neurons, into each of thecells, and isolating the fluorescence-emitting cells.

In its second aspect, the invention of this application provides cellsunder culture condition, which is enriched and isolated by the method ofthe first aspect of the invention.

In its third aspect, the invention of this application provides a methodfor identifying dopaminergic neurons alive, which comprises introducinga reporter nucleic acid molecule that expresses a fluorescent proteinunder control of the promoter/enhancer of a gene that is expressed indopaminergic neurons, into each of the cells, and measuring thefluorescence distribution among these populations of the cells.

In its fourth aspect, the invention of this application provides amethod for identifying a factor which induce the cells that have anability to differentiate into dopaminergic neurons, into dopaminergicneurons, the method comprising introducing a reporter nucleic acidmolecule that expresses a fluorescent protein under control of thepromoter/enhancer of a gene that is expressed in dopaminergic neurons,into cells, incubating the cells with a candidate substance, anddetermining whether the candidate substance is the dopaminergicneurons-inducing factor by using the fluorescence of the cells as anindicator.

In the methods of the first and third aspects of the invention, thefollowing are preferred embodiments:

the gene that is expressed in dopaminergic neurons is a tyrosinehydroxylase gene;

the fluorescent protein is a green fluorescent protein;

the cells are derived from brain;

the cells are ES cells;

the cells are derived from marrow mesenchymal cells;

the cells are derived from human;

each of the cells is introduced with a recombinant vector having thereporter nucleic acid molecule, and/or

the cells are derived from an animal or its progeny which is obtainedthrough the ontogenic development of non-human totipotent cell intowhich the reporter nucleic acid molecule is introduced.

In another preferred embodiment of the first aspect of the invention,the fluorescence-emitting cells are enriched and isolated by the use ofa cell sorter.

Preferred embodiments of the fourth aspect of the invention are thefollowing:

the gene that is expressed in dopaminergic neurons is a tyrosinehydroxylase gene;

the fluorescent protein is a green fluorescent protein;

the cells that have an ability to differentiate into dopaminergicneurons are derived from the brain;

the cells that have an ability to differentiate into dopaminergicneurons are ES cells;

the cells that have an ability to differentiate into dopaminergicneurons are marrow interstitial cells;

the cells that have an ability to differentiate into dopaminergicneurons are derived from human;

the cells that have an ability to differentiate into dopaminergicneurons are introduced with a recombinant vector having the reporternucleic acid molecule; and/or

the cells that have an ability to differentiate into dopaminergicneurons are derived from an animal or its progeny which is obtainedthrough the ontogenic development of non-human totipotent cell intowhich the reporter nucleic acid molecule is introduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of FACS analysis of dispersions of cellsobtained from fetal, TH-EGFP transgenic mouse mesencephalon.

FIG. 2 shows the results of analysis of all cells (A) and FACS-sortedcells (B) in cell dispersions for GFP and TH gene expression.

THE BEST MODE FOR CARRYING OUT THE INVENTION

The first invention is a method that is characterized by the inductionof a reporter nucleic acid molecule, which expresses a fluorescentprotein under control of the promoter/enhancer of a gene that isexpressed in dopaminergic neurons, into various type of animal-derivedcell, and the isolation of the fluorescence-emitting cells from thecells.

The reporter nucleic acid molecule to be introduced into the cells is afusion gene which consists of a DNA sequence encoding thepromoter/enhancer of a gene that is expressed in dopaminergic neuronsand a fluorescent protein-encoding DNA sequence in its downstream.

For the promoter/enhancer of the gene that is expressed in dopaminergicneurons, herein employable is the promoter sequence of a tyrosinehydroxylase (TH) gene of various kinds of animals, and especiallypreferred is the promoter of a rat TH gene. The rat TH gene promotersequence was registered as GenBank Accession No. AF069036. For useherein, it may be obtained by screening the rat genome library, using aprobe constructed on the basis of the known sequence; or it may beobtained through PCR using synthetic primers.

For the fluorescent protein, herein usable is any ofscyphomedusa-derived green fluorescent protein or sea anemone-derivedred fluorescent protein (RFP). Especially preferred are GFP and GFPderivatives (e.g., those described in Current Biology 6(2): 178-182,1996). For the GFP-encoding polynucleotide, its cDNA is known (Gene111(2): 229-233, 1990; GenBank No. M62654). Clones of EGFP cDNA (EGFPPoly(A), from Clontech) are also available.

For the cells into which such a reporter nucleic acid molecule isintroduced, differentiated neural cells derived from brains of animalsincluding human can be used. Also usable are dopaminergic neurons whichare in-vitro differentiated and induced from neural stem cells, EScells, marrow mesenchymal cells or the like having the differentiationpotency into dopaminergic neurons. For inducing the undifferentiatedcells of these types into dopaminergic neurons, employable are any knownmethods (for example, for neural stem cells, referred to is Nat.Neurosci., 1: 290-295, 1998; and for ES cells, referred to are Nat.Biotechnol., 18: 675-679, 2000, and Neuron 28: 31-40, 2000).

For introducing such a reporter nucleic acid molecule into cells,employable is a method of introducing an expression vector having thereporter nucleic acid molecule insert therein, into each cell culture.The expression vector may be a plasmid vector for expression in animalcells. For introducing such a plasmid vector into cells, employable isany of an electroporation method, a calcium phosphate method, a liposomemethod or a DEAE-dextran method. Also employable is a method ofinfecting cells with a viral vector such as an adenoviral vector.

In case where the method is directed to non-human animals, transgenicanimals having the intended reporter nucleic acid molecule introducedthereinto may be produced, and their cells thus having the reporternucleic acid molecule may be used herein. Such transgenic animals may beproduced in any known methods (for example, as in Proc. Natl. Acad.Sci., USA 77, 7380-7384, 1980). The non-human transgenic animals havethe reporter nucleic acid molecule in all their somatic cells. Fromthem, therefore, the tissue in the central nervous system is taken out,and the cells that emit a fluorescent signal are isolated from it. Inthat manner, a large amount of the intended dopaminergic neurons can beobtained.

For enrichment and isolation of the dopaminergic neurons from the cellsinto which the intended reporter nucleic acid molecule has beenintroduced according to any of the methods mentioned above, the cellsthat emit the fluorescent signal may be separated one after another fromthe cultured cells by the use of a fluorescent microscope. However, forgreatly increasing the work efficiency, it is preferable to use a cellsorter (e.g., fluorescence activated cell sorter: FACS). Using such acell sorter, the intended dopaminergic neurons can be automaticallyenriched and isolated.

The method of the third aspect of the invention is for visualizing andidentifying the dopaminergic neurons alive that exist within a mixedcell population. The method comprises introducing the above-mentionedreporter nucleic acid molecule into each of cells, and measuring thefluorescence distribution in the cells. In this, the materials and themethods of nucleic acid molecule introduction into cells may bebasically the same as those in the first aspect of the invention. Thecells into which the reporter nucleic acid molecule has been introducedare observed with a microscope, and the dopaminergic neurons therein canbe visualized and identified by the fluorescence distribution within thecells.

The method of the fourth aspect of the invention is for identifying adopaminergic neurons-inducing factor. This comprises introducing anintended reporter nucleic acid molecule into cells that have the potencyto differentiate into dopaminergic neurons, incubating the cells with acandidate substance, and determining whether the candidate substance isa dopaminergic neurons-inducing factor by using the fluorescence of thecells as an indicator. In this, the cells that have the potency todifferentiate into dopaminergic neurons may be any of neural stem cells,ES cells, marrow mesenchymal cells or the like. The same reporternucleic acid molecule as in the first aspect of the invention mentionedabove is introduced into these undifferentiated cells, and a candidatesubstance is added to the cell culture medium. Whether the candidatesubstance induces the undifferentiated cells into dopaminergic neuronscan readily be confirmed in the same manner as in the second aspect ofthe invention mentioned above.

The invention of this application is described more in detail and moreconcretely with reference to the following Examples, which, however, arenot intended to restrict the scope of the invention.

EXAMPLES

1. Preparation of Transgenic Mouse

Constructed was a vector (RTH-GFP) that expresses GFP under the controlof the promoter sequence of a rat TH gene. Concretely, the upstream10-kb promoter sequence of a rat TH gene that is known to be expressedspecifically in dopaminergic neurons (Mol. Brain Res., 27: 281-289,1994; Mol Cells, 7: 394-398, 1997), was introduced into the upstream ofEGFP cDNA (from Clontech) to construct a recombinant vector. Next, therecombinant vector was cleaved to be linear, and this was injected intothe pronucleus of a zygote derived from an F1 mouse of C57BL/6J mouseand DBA/2J mouse. The gene-introduced zygote was transplanted into theoviduct of a surrogate mother in an ordinary manner, in which it grewinto an individual, TH-EGFP-transgenic mouse.

2. Preparation of Cell Dispersion

The male TH-EGFP mouse was mated with a wild-type mouse. From the 12-dayfetus taken out of it, the ventral mesencephalon was taken out. Thistissue was processed in a solution of trypsin/EDTA, and its cells weredispersed through pipetting. The cells were cultured for 24 hours, andthen reacted with an anti-TH antibody and a Texas Red-labeled secondaryantibody to analyze them. The result confirmed that about more than halfof the GFP-positive antibodies are TH-positive dopaminergic neurons.

3. Enrichment and Isolation of Dopaminergic Neurons with Cell Sorter

Propidium iodide was added to the cell dispersion prepared in the above2, and this was passed through a nylon mesh to remove the non-digestedtissue debris from it. Then, this was analyzed with an FACS Vantage(from Vector Dickinson). As in FIG. 1 showing the result, 7% cells inthe cell dispersion gave a fluorescent signal.

Next, the propidium iodide-negative, GFP-emitting cells (living cells)were collected in a test tube. These were applied to a cover glass, andtested for the reactivity to the antibodies in the same manner as in theabove 2. As in FIG. 2(B) showing the result, almost all the cells arepositive to GFP and to TH. This means that the cells are dopaminergicneurons.

4. The cells obtained in the above 3 were transplanted in parkinsonismmodel rats with 6-OHDA administered thereto. After 5 weeks, the ratswere checked for rotation behaviors to be caused by amphetamineadministered thereto. For their disease symptoms, all the rats weresignificantly improved.

INDUSTRIAL APPLICABILITY

As described in detail hereinabove, the invention of this applicationprovides a method for enrichment and/or isolation of dopaminergicneurons from cells consisting of different types of cells, and providesthe dopaminergic neurons that are enriched to a high purity according tothe method. The cells are useful not only for the material (cells fortransplantation) for treatment of human Parkinson's disease, etc., butalso for analyzing the causes and the symptoms of the disease and fordeveloping the therapeutic techniques and the medicines for the disease.In addition, the invention of this application also provides a methodfor visualizing and identifying dopaminergic neurons alive, andprovides, based on it, a method for identifying the factor thatparticipates in induction of cells to differentiate into dopaminergicneurons. These methods make it possible to efficiently obtain graftcells for Parkinson's disease and others from undifferentiated cells.Further, the dopaminergic neurons-differentiating and inducing factor isuseful for development of novel drugs for treatment.

1. A method for enriching and/or isolating dopaminergic neurons from anon-human transgenic animal or its progeny, which comprises: introducinga recombinant vector comprising a reporter nucleic acid molecule thatexpresses a fluorescent protein under control of the promoter/enhancerof a tyrosine hydroxylase gene of the non-human animal into a fertilizedegg of the non-human animal, developing the non-human fertilized eggwhose genome comprises the reporter nucleic molecule into a non-humantransgenic animal by transfer to a surrogate mother, and isolating cellsthat emit a fluorescent signal from a plurality of cells of thenon-human transgenic animal or its progeny whose genome comprises thereporter nucleic acid molecule, wherein the fluorescence-emitting cellsare dopaminergic neurons.
 2. The method as claimed in claim 1, whereinthe fluorescent protein is a green fluorescent protein.
 3. The method asclaimed in claim 1, wherein the plurality of cells are derived frombrain of the non-human transgenic animal or its progeny.
 4. The methodas claimed in claim 1, wherein the plurality of cells are derived frommarrow mesenchymal cells of the non-human transgenic animal or itsprogeny.
 5. The method as claimed in claim 1, wherein thefluorescence-emitting cells are enriched and isolated by the use of acell sorter.
 6. A cell culture comprising cells that have been enrichedand isolated by the method of claim
 1. 7. A method for identifying livedopaminergic neurons from a non-human transgenic animal or its progeny,which comprises: introducing a recombinant vector comprising a reporternucleic acid molecule that expresses a fluorescent protein under controlof the promoter/enhancer of a tyrosine hydroxylase gene of the non-humananimal into a fertilized egg of the non-human animal, developing thenon-human fertilized egg whose genome comprises the reporter nucleicacid molecule into a non-human transgenic animal by transfer to asurrogate mother, and measuring the fluorescence distribution within aplurality of cells obtained from the non-human animal or its progenywhose genome comprises the reporter nucleic acid molecule, wherein thefluorescence is an indicator of live dopaminergic neurons.
 8. The methodas claimed in claim 7, wherein the fluorescent protein is a greenfluorescent protein.
 9. The method as claimed in claim 7, wherein theplurality of cells are derived from brain of the non-human transgenicanimal or its progeny.
 10. The method as claimed in claim 7, wherein theplurality of cells are derived from marrow mesenchymal cells of thenon-human transgenic animal or its progeny.
 11. A method for identifyinga factor which induces cells to differentiate into dopaminergic neurons,which comprises: introducing a recombinant vector comprising a reporternucleic acid molecule that expresses a fluorescent protein under controlof the promoter/enhancer of a tyro sine hydroxylase gene of thenon-human animal into a fertilized egg of the non-human animal,developing the non-human fertilized egg whose genome comprises thereporter nucleic acid molecule into a non-human transgenic animal bytransfer to a surrogate mother, incubating cells obtained from thenon-human transgenic animal or its progeny whose genome comprises thereporter nucleic acid molecule with a candidate substance, anddetermining whether the candidate substance is the dopaminergicneuron-inducing factor by identifying cells that emit a fluorescentsignal, greater than background fluorescence to indicate the presence ofthe dopaminergic neuron-inducing factor.
 12. The method as claimed inclaim 11, wherein the fluorescent protein is a green fluorescentprotein.
 13. The method as claimed in claim 11, wherein the cells thathave an ability to differentiate into dopaminergic neurons are neuralstem cells of the non-human transgenic animal or its progeny.
 14. Themethod as claimed in claim 11, wherein the cells that have an ability todifferentiate into dopaminergic neurons are marrow mesenchymal cells ofthe non-human transgenic animal or its progeny.
 15. A method forenriching and/or isolating dopaminergic neurons from a transgenic mouseor its progeny, which comprises: introducing a recombinant vectorcomprising a reporter nucleic acid molecule that expresses a fluorescentprotein under control of the promoter/enhancer of a tyro sinehydroxylase gene of the mouse or the promoter/enhancer of a tyrosinehydroxylase gene obtained from a rat into mouse ES cells, developing themouse ES cells whose genome comprises the reporter nucleic acid moleculeinto a transgenic mouse by introducing the ES cells into a blastocystand transferring the blastocyst to a surrogate mother, and isolatingcells that emit a fluorescent signal from a plurality of cells obtainedfrom the transgenic mouse or its progeny whose genome comprises thereporter nucleic acid molecule, wherein the fluorescence-emitting cellsare dopaminergic neurons.
 16. The method as claimed in claim 15, whereinthe fluorescent protein is a green fluorescent protein.
 17. The methodas claimed in claim 15, wherein the fluorescence-emitting cells areenriched and isolated by the use of a cell sorter.
 18. A cell culturecomprising the cells that have been enriched and isolated by the methodof claim
 15. 19. A method for identifying live dopaminergic neurons froma transgenic mouse or its progeny, which comprises: introducing arecombinant vector comprising a reporter nucleic acid molecule thatexpresses a fluorescent protein under control of the promoter/enhancerof a tyrosine hydroxylase gene of the mouse or the promoter/enhancer ofa tyrosine hydroxylase gene obtained from a rat into mouse ES cells,developing the mouse ES cells whose genome comprises the reporternucleic acid molecule into a transgenic mouse by introducing the EScells into a blastocyst and transferring the blastocyst to a surrogatemother, and measuring the fluorescence distribution within a pluralityof cells obtained from the transgenic mouse or its progeny whose genomecomprises the reporter nucleic acid molecule, wherein the fluorescenceis an indicator of live dopaminergic neurons.
 20. The method as claimedin claim 19, wherein the fluorescent protein is a green fluorescentprotein.